The delivery of RNAi triggers and other substantially cell membrane impermeable compounds into a living cell is highly restricted by the complex membrane system of the cell. Drugs used in antisense, RNAi, and gene therapies are relatively large hydrophilic polymers and are frequently highly negatively charged. Both of these physical characteristics severely restrict their direct diffusion across the cell membrane. For this reason, the major barrier to RNAi trigger delivery is the delivery of the RNAi trigger across a cell membrane to the cell cytoplasm or nucleus.
Numerous transfection reagents have also been developed that achieve reasonably efficient delivery of polynucleotides to cells in vitro. However, in vivo delivery of polynucleotides using these same transfection reagents is complicated and rendered ineffective by in vivo toxicity, adverse serum interactions, and poor targeting. Transfection reagents that work well in vitro, cationic polymers and lipids, typically form large cationic electrostatic particles and destabilize cell membranes. The positive charge of in vitro transfection reagents facilitates association with nucleic acid via charge-charge (electrostatic) interactions thus forming the nucleic acid/transfection reagent complex. Positive charge is also beneficial for nonspecific binding of the vehicle to the cell and for membrane fusion, destabilization, or disruption. Destabilization of membranes facilitates delivery of the substantially cell membrane impermeable polynucleotide across a cell membrane. While these properties facilitate nucleic acid transfer in vitro, they cause toxicity and ineffective targeting in vivo. Cationic charge results in interaction with serum components, which causes destabilization of the polynucleotide-transfection reagent interaction, poor bioavailability, and poor targeting. Membrane activity of transfection reagents, which can be effective in vitro, often leads to toxicity in vivo.
For in vivo delivery, the vehicle (nucleic acid and associated delivery agent) should be small, less than 100 nm in diameter, and preferably less than 50 nm. Even smaller complexes, less that 20 nm or less than 10 nm would be more useful yet. Delivery vehicles larger than 100 nm have very little access to cells other than blood vessel cells in vivo. Complexes formed by electrostatic interactions tend to aggregate or fall apart when exposed to physiological salt concentrations or serum components. Further, cationic charge on in vivo delivery vehicles leads to adverse serum interactions and therefore poor bioavailability. Interestingly, high negative charge can also inhibit targeted in vivo delivery by interfering with interactions necessary for targeting, i.e. binding of targeting ligands to cellular receptors. Thus, near neutral vehicles are desired for in vivo distribution and targeting. Without careful regulation, membrane disruption or destabilization activities are toxic when used in vivo. Balancing vehicle toxicity with nucleic acid delivery is more easily attained in vitro than in vivo.
Rozema et al., (U.S. Patent Publications 20080152661, 20110207799, 20120165393, and 20120172412) developed conjugates suitable for in vivo delivery of polynucleotides. These conjugates featured reversible regulation of membrane disruptive activity of a membrane active polyamine using reversible physiologically labile masking. Using uncharged galactose or cholesterol as targeting ligands, Rozema et al. have shown in vivo delivery of polynucleotides to hepatocytes using these conjugates. Adaptation of these conjugates to target RNAi triggers to cancer cells would provide another therapeutic in the fight against cancer.
Integrins are a group of cell surface glycoproteins which mediate cell adhesion. Integrins are heterodimers composed of α and β polypeptide subunits. Currently eleven different α subunits and six different β subunits have been identified. The various α subunits combine with various β subunits to form distinct integrins. The αvα3 integrin (vitronectin receptor) has been shown to play a role in tumor metastases, solid tumor growth (neoplasia), and tumor angiogenesis. The integrin αvβ3 plays an important role in angiogenesis. It is expressed on tumoral endothelial cells as well as on some tumor cells. Seftor et al. (Proc. Natl. Acad. Sci. USA, Vol. 89 (1992) 1557-1561), for example, have shown a role for αvβ3 integrin in melanoma cell invasion. Brooks et al. (Cell, Vol. 79 (1994) 1157-1164) demonstrated that systemic administration of αvβ3 antagonists caused dramatic regression of various histologically distinct human tumors.
Tumor cell expression of the integrins αvβ3 is correlated with disease progression in various tumor types. αvβ3 integrin is widely expressed on blood vessels of human tumor biopsy samples but not on vessels in normal tissues. In breast cancer, overexpression of αvβ3 integrin is associated with bone metastasis and induces increased tumor growth and invasion in response to osteopontin. In glioblastoma, αvβ3 integrin is overexpressed at the invasive margins of the tumor and levels of fibronectin are increased, which is associated with enhanced cell motility and apoptosis resistance. In pancreatic tumor, the increased expression of αvβ3 integrin is associated with increased activation of MMP-2 and lymph node metastasis. In prostate carcinoma cell, αvβ3 integrin is expressed resulting in metastasis to bone because of an association between integrins and processes of attachment and migration involving laminin, fibronectin, and osteopontin.
αvβ3 integrins bind to a number of Arg-Gly-Asp (RGD) containing matrix macromolecules. The RGD peptide sequence has been linked to various other compounds to provide αvβ3 integrin binding. Therefore, RGD peptides have been examined for targeting of compounds to αvβ3 integrin positive tumors. However, in addition to relatively low affinity, many RGD peptides are also relatively non-selective for RGD-dependent integrins. For example, most RGD peptides which bind to αvβ3 also bind to αvβ5, αvα1, and αIIbβ3 integrins.